The human central CB1 and peripheral CB2 cannabinoid receptors were expressed in Escherichia coli as fusion proteins with the maltose-binding protein at their amino-termini and a hexa-histidine/Flag ...tag at their carboxyl-termini. Western blot analysis of the expressed proteins revealed considerable degradation of the CB1 fusion, which failed to bind either the cannabinoid agonist CP 55,940 or the CB1-specific antagonist SR 141716A. In contrast, the CB2 fusion was well-expressed and bound several cannabinoids with affinities comparable to those observed in mammalian expression systems.PUBLICATION ABSTRACT
Amino acid sequencing of peptides obtained after proteolytic hydrolysis of Aspergillus flavus urate oxidase (uricase) permitted the design of oligodeoxynucleotide probes that were used to obtain 1.2- ...and 5-kilobase pair DNA fragments from A. flavus cDNA and genomic libraries, respectively. The cDNA fragment contained the entire coding region for uricase, and comparison with the genomic fragment revealed the presence of two short introns in the coding region of the gene. A. flavus uricase has around 40% overall identity with uricases from higher organisms but with many conserved amino acids. Hitherto highly conserved consensus patterns found in other uricases were found to be modified in the A. flavus enzyme, notably the sequence Val-Leu-Lys-Thr-Thr-Gln-Ser near position 150, which in the filamentous fungus is uniquely modified to Val-Leu-Lys-Ser-Thr-Asn-Ser. Silent mutations were introduced by cassette mutagenesis near the 5'-extremity of the coding sequence in order to conform with Escherichia coli codon usage, and the uricase was expressed in the E. coli cytoplasm in a completely soluble, biologically active form.
We hypothesized that charge–charge interactions may be important for the binding of the human cholecystokinin type 1 (CCK
1) receptor-specific non-peptide full agonist SR 146131, ...(2-4-(4-chloro-2,5-dimethoxyphenyl)-5-(2-cyclohexyl-ethyl)-thiazol-2-ylcarbamoyl-5,7-dimethyl-indol-1-yl-1-acetic acid), the competitive antagonist SR 27897, (1-2-(4-(2-chlorophenyl)thiazol-2-yl) aminocarbonyl indoyl acetic acid) and the natural octapeptide CCK-8S to the CCK
1 receptor. Alanine replacement studies of positively charged residues in the extracellular domains of the receptor showed that only the R336A mutation affected SR 146131 potency of mutated receptors transiently expressed in monkey kidney epithelial COS-7 cells. Two residues, Lys
115 and Lys
187, were implicated in SR 27897 binding. Only the replacement of Lys
115, Arg
197 and Arg
336 significantly affected CCK-8S binding or activity. These results clearly indicated the importance of certain charged residues, but not others, in SR 146131, SR 27897 and CCK-8S binding. Furthermore, although these molecules probably occupy different binding sites on the CCK
1 receptor, we show that a small non-peptide agonist, SR 146131, can stimulate the dual signaling pathways mediated by the CCK
1 receptor.
1-2-(4-(2-Chlorophenyl)thiazol-2-yl) aminocarbonyl indoyl acetic acid (SR 27897) is an effective CCK
1 receptor antagonist, while the structurally related molecule ...2-4-(4-chloro-2,5-dimethoxyphenyl)-5-(2-cyclohexyl-ethyl)-thiazol-2-ylcarbamoyl-5,7-dimethyl-indol-1-yl-1-acetic acid (SR 146131) is a highly potent and specific agonist for the same receptor. To discover how the two molecules interact with the human cholecystokinin (CCK) CCK
1 receptor, we have carried out binding and activity studies with 33-point mutated receptors. Only six mutants showed altered
3HSR 27897 binding properties, Lys
115, Lys
187, Phe
198, Trp
209, Leu
214 and Asn
333. In contrast, numerous mutations throughout the receptor either reduced SR 146131 agonist potency, Phe
97, Gly
122, Phe
198, Trp
209, Ile
229, Asn
333, Arg
336 and Leu
356 or increased it, Tyr
48, Cys
94, Asn
98, Leu
217 and Ser
359. Only mutations of Phe
198, Trp
209 and Asn
333 affected both SR 27897 and SR 146131 binding or activity. The collated information was used to construct molecular models of SR 27897 and SR 146131 bound to the human CCK
1 receptor. The clear difference in the binding sites of SR 27897 and SR 146131 offers a molecular explanation for their contrasting pharmacological characteristics.
The 18 kDa peripheral benzodiazepine receptor (PBR) can be labelled by benzodiazepines, such as Ro5-4864, and isoquinoline carboxamides such as PK11195. These two compounds are reversible competitive ...inhibitors of each other. However, while the binding affinity of Ro5-4864 varies enormously across species, PK11195 always displays high affinity, suggesting that their binding domains are overlapping but not identical. We report here that recombinant human and bovine PBR produced in yeast, a microorganism devoid of endogenous PBR, can be labelled with
3HPK11195, but only the human receptor can be labelled with
3HRo5-4864. Furthermore, we identified, through the binding analysis of human-bovine chimaeric receptors, a region near the C-terminal end of the PBR, with only five non-conserved amino acids between human and bovine sequences, as responsible for the difference in high affinity binding of Ro5-4864 to the two receptors.
A new highly specific, potent non-peptide agonist for the cholecystokinin subtype 1 receptor (CCK
1), SR 146131 ...(2-4-(4-chloro-2,5-dimethoxyphenyl)-5-(2-cyclohexyl-ethyl)-thiazol-2-ylcarbamoyl-5,7-dimethyl-indol-1-yl-1-acetic acid) was recently described Bignon, E., Bachy, A., Boigegrain, R., Brodin, R., Cottineau, M., Gully, D., Herbert, J.-M., Keane, P., Labie, C., Molimard, J.-C., Olliero, D., Oury-Donat, F., Petereau, C., Prabonneaud, V., Rockstroh, M.-P., Schaeffer, P., Servant, O.Thurneyssen, O., Soubrié, P., Pascal, M., Maffrand, J.-P., Le Fur, G., 1999. SR 146131: a new, potent, orally active and selective non-peptide cholecystokinin subtype I receptor agonist: I. In vitro studies. J. Pharmacol. Exp. Ther. 289, 742–751. From binding and activity assays with chimeric constructs of human CCK
1 and the cholecystokinin subtype 2 receptor (CCK
2) and receptors carrying point mutations, we show that Leu
356, situated in transmembrane domain seven in the CCK
1 receptor, is a putative contact point for SR 146131. In contrast, Leu
356 is probably not in contact with the CCK
1 receptor specific antagonist SR 27897 (1-2-(4-(2-chlorophenyl)thiazol-2-yl)aminocarbonyl indoylacetic acid), a compound structurally related to SR 146131, since its replacement by alanine, histidine or asparagine gave receptors having wild-type CCK
1 receptor SR 27897 binding affinity. Previous mutational analysis of His
381, the cognate position in the rat CCK
2 receptor, had implicated it as being involved in subtype specificity for SR 27897, results which we confirm with corresponding mutations in the human CCK
2 receptor. Moreover, binding and activity assays with the natural CCK receptor agonist, CCK-8S, show that CCK-8S is more susceptible to the mutations in that position in the CCK
1 receptor than in the CCK
2 receptor. The results suggest different binding modes for SR 27897, SR 146131 and CCK-8S in each CCK receptor subtype.
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